U.S. patent number 4,617,275 [Application Number 06/653,861] was granted by the patent office on 1986-10-14 for reagent for blood analysis.
This patent grant is currently assigned to Toa Medical Electronics Co., Ltd.. Invention is credited to Noriaki Matsuda, Etsuro Shinkai.
United States Patent |
4,617,275 |
Matsuda , et al. |
October 14, 1986 |
Reagent for blood analysis
Abstract
A reagent for blood analysis comprises, as a lysing reagent for
leukocyte count, tetradecyltrimethylammonium bromide and/or
hexadecyltrimethylammonium chloride, dodecyltrimethylammonium
chloride, and citric acid. This reagent is characterized by showing
three-peaks fractionation of leukocytes in measuring by means of an
automatic blood-analysis instrument. The lysing reagent is used
together with a diluent comprising boric acid buffer solution,
ethylenediaminetetraacetic acid and (2-pyridylthio-1-oxide)
sodium.
Inventors: |
Matsuda; Noriaki (Kakogawa,
JP), Shinkai; Etsuro (Akashi, JP) |
Assignee: |
Toa Medical Electronics Co.,
Ltd. (Kobe, JP)
|
Family
ID: |
16145475 |
Appl.
No.: |
06/653,861 |
Filed: |
September 24, 1984 |
Foreign Application Priority Data
|
|
|
|
|
Sep 29, 1983 [JP] |
|
|
58-183995 |
|
Current U.S.
Class: |
436/10; 436/17;
436/18 |
Current CPC
Class: |
G01N
33/5094 (20130101); G01N 33/56972 (20130101); Y10T
436/101666 (20150115); Y10T 436/108331 (20150115); Y10T
436/107497 (20150115) |
Current International
Class: |
G01N
33/569 (20060101); G01N 33/50 (20060101); G01N
033/48 () |
Field of
Search: |
;252/408 ;424/2-3
;436/17,18,8,10,15 ;435/2 ;422/61 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Terapane; John F.
Assistant Examiner: Wallen; T. J.
Attorney, Agent or Firm: Stevens, Davis, Miller &
Mosher
Claims
What is claimed is:
1. A reagent for preparing cells for blood analysis comprising, as
a lysing reagent for leukocyte count measurement,
dodecyltrimethylammonium chloride; citric acid; and at least one
member selected from the group consisting of
tetradecyltrimethylammonium bromide and hexadecyltrimethylammonium
chloride.
2. The reagent of claim 1 wherein said lysing reagent comprises 2.5
to 3.5 parts by weight of hexadecyltrimethylammonium chloride, 26
to 31.5 parts by weight of dodecyltrimethylammonium chloride and
0.01 to 0.02 parts by weight of citric acid.
3. The reagent of claim 1 wherein said lysing reagent comprises 1.6
to 2 parts by weight of hexadecyltrimethylammonium chloride, 1.6 to
2 parts by weight of tetradecyltrimethylammonium bromide, 26 to
31.5 parts by weight of dodecyltrimethylammonium chloride and 0.01
to 0.02 parts by weight of citric acid.
4. The reagent of claim 1 wherein said lysing reagent comprises 3
to 4 parts by weight of tetradecyltrimethylammonium bromide, 26 to
31.5 parts by weight of dodecyltrimethylammonium chloride and 0.01
to 0.02 parts by weight of citric acid.
Description
BACKGROUND OF THE INVENTION
This invention concerns a reagent for blood analysis especially
suitable for use in measurement of leukocyte count in a blood by
means of an automatic blood-analysis instrument.
Ingredients of blood are closely related with a whole organization
or internal organs of human body. Therefore, analytical data of
blood are important as an index of diagnosis or treatment.
Especially, leukocytes of blood include many useful informations
for diagnosis or treatment of organism, since they show a rapid
response to variations of organism conditions.
Up to now, leukocyte classification which is carried out by
classifying leukocytes into 5 to 8 kinds and counting them has been
practiced as an inspection of leukocytes. However, since this
inspection depends on visual count by a microscope, the inspection
including pretreatment takes comparatively many hours. Further, the
determination of visual count demands the expert.
On the other hand, operations of inspection based on leukocyte
classification have been partially automated by introducing the
pattern-recognition technology of a computer. However, even if the
partial automation is possible, the final determination of the
inspection is depended on the expert, and it takes many hours for
the inspection. Further, large-sized and expensive instruments are
demanded for the inspection.
Recently, the automatic blood-analysis instrument which is equipped
with a mechanism for counting blood corpuscles such as leukocyte,
red corpuscles, platelets and so on has developed to accomplish
simplicity and rapidity of the blood analysis. The blood count is
carried out by an electric resistance system as shown in FIG. 6.
According to this system, the blood is diluted with a diluent 1,
and therefore the blood corpuscles 2 are dispersed in the diluent
1. When negative pressure is added in a detector 3, the dispersed
blood corpuscles 2 are sucked in the detector 3 through a small
hole 4. In that case, outer and inner sides of the detector 3 are
provided with outer and inner electrodes 5, 6, respectively, so
that a certain current is flowed from the outer electrode 5 to the
inner electrode 6. When the blood corpuscles 2 of which the
electric resistance is extremely larger than that of the
electrolyte (the diluent 1) are passed through the small hole 4,
the resistance between both electrodes is changed. Such a change of
resistance is taken out as a signal to count the blood corpuscles
such as leukocytes.
Before making leukocyte count by means of the electric resistance
system, the blood is diluted to a certain dilution magnification,
and a certain amount of a lysing reagent for leukocyte count is
added. The lysing reagent solves erythrocytes and cytoplasm of
leukocytes, and leaves the nuclei to be counted. The nuclei is
passed through the small hole 4 of the detector 4.
However, in the traditional practice for counting leukocytes, since
a surface active reagent having the strong lyse-force is used as a
lysing reagent, all of leukocytes to be counted arrive at the
neighborhood of contraction limit, so that total count only, i.e.
one-peak fractionation in size distribution as shown in FIG. 7, has
been carried out.
In order to improve the one-peak fractionation and to emphasize the
difference of the particle size inherent in leukocytes, the lysing
reagent capable of slowing the contraction speed of large particles
and rapidly contracting small particles such as lymphoid cells has
been developed. This lysing reagent contains, as a main ingredient,
dodecyltrimethylammonium chloride and tetradecyltrimethylammonium
bromide, and thus accomplishes two-peaks fractionation by means of
a blood corpuscle counter. This fractionation if called as a
discrimination histogram of the populations of lymphoid and myeloid
cells or a leukocyte volume histogram. A pattern of two-peaks
fractionation is shown in FIG. 8.
Such a blood analysis practiced by the automatic blood-analysis
instrument has an advantage that other measuring items, such as
erythrocyte number, hemoglobin amount, hematocrit value, platelet
number and so on, as well as leukocyte, can be simultaneously
measured in a short time. However with respect to leukocyte count,
two-peaks fractionation was unsatisfactory in comparison with the
practical 6 items classification. Thus, it has been demanded to
raise the fractionation number.
SUMMARY OF THE INVENTION
The present invention relates to a reagent for blood analysis
capable of making three-peaks fractionation in an automatic
blood-analysis instrument.
This reagent for blood analysis comprises, as a lysing reagent for
leukocyte count, tetradecyltrimethylammonium bromide and/or
hexadecyltrimethylammonium chloride, dodecyltrimethylammonium
chloride and citric acid.
Furthermore, this reagent for blood analysis comprises a
combination of a lysing reagent and a diluent; the lysing reagent
comprising tetradecyltrimethylammonium bromide and/or
hexadecyltrimethylammonium chloride, dodecyltrimethylammonium
chloride and citric acid; and the diluent comprising boric acid
buffer solution, ethylenediaminetetraacetic acid and
(2-pyridylthio-1-oxide)sodium.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing size distribution fractioned in three
peaks;
FIG. 2 is a graph showing practically measured size distribution in
accordance with the present invention;
FIGS. 3 (A) and (B) are graphs showing the examination results in
Example 2 and Comparative Example 2, respectively;
FIGS. 4 (A) and (B) are graphs showing the examination results in
Example 2 and Comparative Example 2, respectively;
FIGS. 5 (A) and (B) are graphs showing the examination results in
Examples and Comparative Example 3, respectively;
FIG. 6 is an explanatory view showing a mechanism for counting
blood corpuscles in the conventional automatic blood-analysis
instrument;
FIGS. 7 and 8 are graphs showing size distribution having one-peak
fractionation and two-peaks fractionation.
DETAILED DESCRIPTION
In the present invention, a combination of each quaternary ammonium
salt consisting of tetradecytrimethylammonium bromide and/or
hexadecyltrimethylammonium chloride, and dodecytrimethylammonium
chloride can shrink eosinophils of granulocytes and monocytes in
some degree more than lymphocytes in lymphoid/myeloid population,
and as a result, forms three-peaks fractionation of leukocytes as
shown in FIG. 1. In FIG. 1, Peaks A, B, C indicate normal
lymphocytes, eosinophil monocytes and other granulocytes,
respectively. In this connection, peak D in two-peaks fractionation
shown in FIG. 8 mainly indicates normal lymphocytes, and peak E
indicates other components of leukocytes.
One or mixture of the above-mentioned tetradecyltrimethylammonium
bromide (hereinafter referred to as "MTAB") and
hexadecyltrimethylammonium chloride (hereinafter referred to as
"CTAC") are preferably contained in the lysing reagent. In that
case, to the aqueous solution in which the above ingredients are
solved, citric acid and dodecyltrimethylamonium chloride
(hereinafter referred to as "LTAC") are added to form the lysing
reagent.
Effect of each ingredient is described below. LTAC can make
differences to the contraction rate corresponding to each particle
diameter, so that lymphocytes group and granulocytes group are
fractionated individually with a large gap, since the contraction
effect of LTAC weakens against leulocytes under the suitably
arranged concentration. Nevertheless, LTAC has a disadvantage that
ghosts of red blood cells are not shrinked, and are admixed with
small size cells in lymphocytes. In order to eliminate the
disadvantage, citric acid is added. Citric acid is caused to
accelating the contraction of membranes (ghosts of red blood
cells), and inhibits the aggregation of those.
MTAB has a relatively stronger contraction effect than LTAC. On the
other hand, this effect is decreased with the passage of time at a
certain range, and thus MTAB can make two-peaks fractionation of
lymphocytes and granulocytes. But, since both peaks come into
contact with each other, MTAB is used only for one-peak
fractionation by itself. Though CTAC has a relatively stronger
contraction effect than MTAB, CTAC facilitate appearance of a
central peak in three-peaks fractionation due to a tendency that
eosinophil granulocytes and monocytes in granular leukocytes are
more strongly shrinked.
The preferred compositions of the lysing reagent are described
below.
______________________________________ (Ingredient) (% by weight)
______________________________________ (A) A combination of LTAC
and CTAC LTAC 26 to 31.5 CTAC 2.5 to 3.5 Citric acid 0.01 to 0.02
Water 61 to 75 Total 100 (B) A combination of LTAC, MTAB and CTAC
LTAC 26 to 31.5 MTAB 1.6 to 2 CTAC 1.0 to 2 Citric acid 0.01 to
0.02 Water 61 to 75 Total 100 (C) A combination of LTAC and MTAB
LTAC 26 to 31.5 MTAB 3 to 4 Citric acid 0.01 to 0.02 Water 61 to 75
Total 100 ______________________________________
In these combinations (A)to (C), when the amount of LTAC is more
than the above range, the lysing reagent produces an insufficient
fractionation due to contiguity of each peak of lymphatic
leukocytes and other leukocytes. On the other hand, when the amount
of LTAC is less than the above range, the lysing reagent produces
an insufficient fractionation between lymphatic leukocytes and
ghosts of erythrocytes. Also, when the amounts of CTAC nd MTAB to
be added are more than the above range, the lysing reagent produces
an insufficient fractionation between a group of eosiniphil
granulocytes and monocytes and a group of other granulocytes. When
the amounts of CTAC, MTAB and citric acid is less than the above
range, the lysing reagent produces an insufficient fractionation
between lymphocytes and ghosts of erythrocytes.
A method for making leukocyte count by means of the automatic
blood-analysis instrument is described below. The method comprises
diluting the gathered blood in a dilute till a certain dilution,
which generally is 250 times, adding the lysing reagent, and
detecting the size distribution by the electric resistance system
mentioned above after 30 to 45 seconds. The lysing reagent is added
in the ratio of 1 cc., when 2 cc. of the diluent is added to 0.008
cc. of blood sample. FIG. 2 shows the practical size distribution
measured by the above-mentioned method. The size distribution of
leukocytes is changed into signals in the detector, and is
transmitted through an amplification/discrimination circuits and an
interface circuit to a recorder to be recorded by a microcomputer.
As an automatic blood-analysis instrument, "MULTI-ITEMS AUTOMATIC
BLOOD CORPUSCLE COUNTER CC-800" manufactured by TOA MEDICAL
ELECTRONICS CO., LTD. of Japan can be preferably employed. This
counter is designed so as to automate pretreatment for arranging
sample, which includes diluting step for diluting blood in a high
diluted magnification and lysing step for measuring leukocyte and
hemoglobin, and can treat and analyze sample at a rate of
approximately 80 samples per hour.
Dilution of blood is an important step for preparing sample as well
as lysing step, since pH and osmotic pressure of the dilute to be
used have a large influence on blood corpuscles in its form and
change with the passage of time. Accordingly, the dilute should be
arranged to the prescribed pH and osmotic pressure. As to osmotic
pressure, the dilute is arranged to an isotonic solution having the
prescribed concentration of sodium chloride, and dilutes blood
within the range of approximately 200 to 500 times in connection
with the items to be measured. Also, pH buffer solution which is
added for pH arrangement comprises boric acid and its salt. The
conventional dilute which includes phosphoric acid buffer solution
is undesirable in view of raising BOD value of waste liquid and
resulting in a rich nutrition of the river or the lake.
The dilute is common to each measurement in hemoglobin, leukocyte
and erythrocyte, and the dilution is changed every each measurement
purpose. In hemoglobin measurement, blood is diluted to 500 times
in the dilute. Then, lysing reagent is added to the diluted
solution of blood so as to solve erythrocytes sufficiently, and the
amount of hemoglobin is measured by a photoelectric
colorimeter.
In previous measurement of hemoglobin, the lysing reagent and the
dilute to be used has been the same as in leukocyte measurement,
and further a cyanic compound has been added to the lysing reagent
to change hemoglobin into methemoglobin, i.e. oxidation from
Fe.sup.2.sup.30 contained in hemoglobin to Fe.sup.3.sup.30.
However, this method has caused the problem of the treatment of
waste liquid including cianide. Therefore, oxyhemoglobin processes
has been employed instead of the previous process mentioned above.
In oxyhemoglobin process, it is necessary to prevent change of
hemoglobin into methemoglobin, since the existance of methemoglobin
causes errors in measurement.
According to the present invention, in order to prevent the change
of hemoglobin, (2-pyridylthio-1-oxide)sodium is employed as a
preservative in addition to boric acid buffer solution. This
compound has the formula: ##STR1##
Furthermore, EDTA-2K is used as a chelating reagent together with
the above ingredients. Then, all ingredients are solved in a high
quality water which is refined by using reverse-osmosis and active
carbons. The diluent thus refined has 6 to 8 of pH, and 240 to 310
mOsm/kg. H.sub.2 O of osmotic pressure. This diluent is arranged to
a certain magnification of dilution in accordance with each
measuring item, e.g. 250 times in leukocyte measurement, 500 times
in hemoglobin measurement and about 30,000 times in erythrocyte
measurement.
The diluent and the lysing reagent in the present invention
accomplish the count based on three-peaks fractionation, such as
eosinophils, monocytes, lymphocytes and other ingredients. Further,
the present invention can prevent generating an environmental
pollution due to avoiding cyanide and phosphorus. In addition, the
reagent of the present invention facilitate to obtain the valuable
and accurate informations concerning blood without taking the
larger cost than the conventional operation, together with rapidity
and easiness in use of the automatic blood-analysis instrument.
The following Examples are given to illustrate the present
invention.
EXAMPLE 1
Leukocyte count of blood taken from patient 1 was measured by
"MULTI-ITEMS AUTOMATIC BLOOD CORPUSCLE COUNTER CC-800" mentioned
above. In that case, measurement was carried out by electrically
resistant detection system. As a pretreatment, 0.08 ml. of blood
was diluted to 250 times in 2 ml. of a dilute, and then 1 ml. of a
lysing reagent was added to the sample. After 30 seconds, the
corpuscles were passed through a small hole provided in the
detector.
The composition of the lysing reagent employed is an follows:
LTAC--28.5 g
CTAC--3.1 g
Citric acid--0.015 g
Water--68.385 g
The size distribution of leukocytes thus obtained is shown in FIG.
3 (A).
COMPARATIVE EXAMPLE 1
Measurement of leukocytes was carried out in the same manner as
described in Example 1 except that the conventional lysing reagent
was employed against the same blood taken from patient 1. The
result shown in FIG. 3 (B).
EXAMPLE 2
Measurement of leukocytes was carried out in the same manner as
described in Example 1 except that blood taken from patient 2 was
used as a sample, and that the lysing reagent having the following
composition was used. The result is shown in FIG. 4 (A).
LTAC--28.5 g
MTAB--1.8 g
CTAC--1.8 g
Citric acid--0.015 g
Water--67.885 g
COMPARATIVE EXAMPLE 2
Measurement of leukocytes was carried out in the same manner as
described in Example 2 except that the lysing reagent was the same
as that of Comparative Example 1. The result is shown in FIG. 4
(B).
EXAMPLE 3
Measurement of leukocytes was carried out in the same manner as
described in Example 1 except that blood taken from patient 3 was
used as a sample, and that the lysing reagent having the following
composition was used. The result is shown in FIG. 5 (A).
LTAC--28.5 g
MTAB--3.5 g
Citric acid--0.015 g
Water--67.985 g
COMPARATIVE EXAMPLE 3
Measurement of leukocytes was carried out in the same manner as
described in Example 3 except that the lysing reagent was the same
as that of Comparative Example 1. The result is shown in FIG. 5
(B).
As obvious from these results, though the lysing reagents employed
in each Example clearly show three-peaks fractionation, those
employed in each comparative Example show only two-peaks
fractionation.
* * * * *